Sec4 is a small GTPase of the rab family that is associated with the secretory vesicles of yeast. Sec 4 interacts with different sets of proteins in its different nucleotide states. In its GDP bound state it interacts with GD1, a proteins that maintains Sec4 in the cytosol. In its nucleotide free state, Sec4 will interact with DSS4 or with Sec2. Dss4 acts to stimulate dissociation of nucleotides for Sec4, while Sec2, working in conjugation with the actin based cytoskeleton, is needed for the polarized concentration of secretory vesicles at sites of exocytosis. Fst1 is a proteins that interacts with Sec2 to negatively regulate its function. Several newly identified proteins will interact with Sec4 in its GTP bound state. A number of studies are proposed to address the mechanisms by which these proteins work together to control the cycle of Sec4 function and to allow it to fulfill its role in targeting vesicles to specialized regions of the cell surface. 1) We will define the structural domains of Sec2 that allow binding to Sec4, oligomerization, localization to the bud tip and interaction with Fst1. 2) We will determine the mechanism by which Sec2 is localized to the bud tip and identify factors that interact with its localization domain, possibly linking it to the cytoskeleton. 3) We will establish the role of Sec2 in Sec4 function, evaluating its effect on the GDP off rate, the GTP on rate and the interactions with Dss4, Gdi1 and the cytoskeleton. 4) We will analyze the role of two different suppressors of dominant negative alleles of SEC4;SMY1 and a novel gene encoding 27kD protein. 5) We will explore a novel approach to identify a GAP for Sec4 and pursue the function of several Sec4 interacting proteins and a highly conserved protein that interacts with Dss4.